We narrowed to 432 results for: sequence

...assemble large DNA sequences, no restriction enzymes needed Not optimal for joining sequences with a high degree...coding sequences), so enzyme choice is critical. May not support in-frame fusions of coding sequences Sladitschek...the systematic combination of different genetic sequences into plasmid DNA. In high throughput cloning techniques... techniques, although the specific sequences of the genetic elements may differ (e.g., a set of various...the combination of multiple fluorescent protein sequences to develop diverse fluorescent reporters is a ...polyadenylation signals, inducible gene-expression sequences, and more. By combining these components, one ...necessarily suitable for combining endogenous sequences. Table 2: High Throughput Cloning Strategies ...

...CrispyCrunch is how it mutates guide sequences to prevent re-cutting of the sequence inserted by HDR. Although this...algorithm then compares the guide sequence to every possible 23bp sequence (protospacer + PAM) in the target... often because the HDR inserted sequence splits up the guide sequence.) If neither of the above are options...codons in the guide sequence, one by one, from the PAM outwards so that the DNA sequence is altered but the...needed to design guide RNAs, donor templates and sequencing primers for quality control. At such a scale,...requires the additional step of mutating guide sequences in a way that prevents re-cutting but does not...not be enough to prevent re-binding. Lastly, the sequence inserted by HDR may inadvertently re-create a ...

...end-joining (NHEJ) pathway disrupt target gene sequences (thus inactivating genes), while breaks repaired... directed repair (HDR) pathways can modify the sequence of a gene (thus altering its function). HDR is...rational approach to improve the efficiency of sequence replacement by HDR. Understanding Cas9 biophysics...from in vitro studies) to boost the efficiency of sequence replacement in vivo. We discovered that single...from Richardson et al., 2016.   dCas9 enables sequence replacement without cutting Our in vitro results... thus wondered if such a structure could drive sequence replacement in cells. Targeting three dCas9 molecules...molecules to a precisely spaced region allowed sequence replacement rates of approximately 1%. This is...

...sneaky little bit of sequence that you hope is present next to the "perfect" guide sequence for your genome... a Protospacer Adjacent Motif (PAM) sequence. If the gRNA sequence is suitable, but there is no PAM next...typically selected by the ‘quality’ of the gRNA sequence, including propensity for off-targets, secondary...To add insult to injury, the most utilized PAM sequence, that of SpCas9, is NGG. Assuming all nucleotides...Cas enzyme or evolve one that uses a PAM with no sequence constraints, or at least has looser or different...such as Cpf1/Cas12a, which recognizes the PAM sequences of TTTV. While all of these enzymes certainly ...equal; just because an enzyme recognizes a PAM sequence in your site doesn’t mean it’s suitable for your...

... from Addgene’s sequencing reactions (‘Addgene Sequences’).  These sequences are provided in FASTA format... your research! Sequence information: Depositors may provide full or partial sequence information for ...be made available as ‘Depositor Sequences’. Additional partial sequence information is also available from...auto-generate a plasmid map based off of the plasmid sequence and feature information given by our depositors...example) and can be analyzed using Addgene’s Analyze Sequence tool. Excellent, how can I check if the plasmid... own lab.  If depositors have provided primer sequences for use, these are listed.  We also provide a ...a link to our list of common primers used for sequencing. Growth in Bacteria: This section describes how...

...specific DNA sequences and companion endonucleases that recognizes and cleave the same DNA sequence if not ...methyltransferases, methylate DNA at particular sequences to prevent them from being degraded by restriction...with any restriction modification systems, their sequences may overlap with restriction sites, inhibiting...therefore have methylated adenines in any GATC sequences found in the plasmid. When the PCR products are...plasmids which can later be verified by Sanger sequencing. How can I tell if my enzyme will cut? Whether...enzyme is affected by methylation, knowing the sequence of the DNA surrounding your restriction site(s...methylation), the figure below illustrates how sequence visualization software (we used Snapgene here)...

...the DNA and test for sequence complementarity with its guide RNA. If the sequence is recognized, Cas9 ...bases, or why it’s so important to choose a target sequence with a nearby protospacer-adjacent motif (PAM)...recognize PAMs, so it associates with random DNA sequences (Sternberg et al., 2014). The nuclease lobes are...editing requires three major steps: RNA loading, DNA sequence recognition, and DNA cleavage. RNA loading Once... (left) and transparent (right). The RNA guide sequence is extended as a single unpaired strand in the...anti-repeat duplex of the guide RNA, confirming its sequence. This region of the protein varies between Cas9... RNA loading: the structure to interrogate the sequence isn’t in place yet.     Figure 4: S. pyogenes...

...   Step 1: Sequences, Maps, and Files Please provide any full or partial plasmid sequences and any vector...The more sequence data available, the better. We highly encourage you to upload full sequence data whenever...files, etc). We can accept sequence files in any format. Figure 6: Add sequence, maps, and files. Step 2...addgene.org along with your GenBank files, plasmid sequences, and/or maps. Add plasmids to your article After...plasmid with one or more genes, protein coding sequences, or other elements inserted into it. You will ...shRNA A plasmid with one or more gRNA or shRNA sequences cloned into it. gRNAs are used for directing CRISPR...expression of mRNAs with complementarity to the shRNA sequence. Empty backbone A plasmid designed for cloning...

...can worry less about sequence optimality because you generally have fewer sequences to choose from). Finally...location and sequence are of approximately equal importance in design – an optimized sequence will do little...modification. Whole-genome sequencing of cells modified by CRISPR indicates that the consequences of off-target activity...or modulation of gene expression. Location and sequence are important considerations for designing your...you target, but it is important that your gRNA sequence is designed to be highly active and reduce off...means there are so few gRNAs to choose from that sequence preferences must largely be ignored. Created...possibilities, picking a gRNA with an optimized sequence is of primary importance (more on this below)....

...introduction of specific sequence changes. To generate a specific sequence change the cell must undergo...CRISPR/Cas9 extensively for the purpose of making sequence-specific changes in human induced pluripotent ...oligodeoxynucleotide (ssODN), which contains the intended sequence changes to be incorporated into the edited genome...our excitement turned into frustration when we sequenced our cells and found how inefficiently our edits... – but avoid NAG) or guide RNA target (“seed”) sequence. The idea was that these “blocking mutations” ...mutations prevent CRISPR from re-cutting the target sequence once the desired edit has been introduced. We ...So if the locus does not allow blocking the PAM sequence (e.g. if it would lead to a missense or nonsense...